A mutational approach will be used to understand how the energy form ATP binding and hydrolysis is converted into myosin's force And motion generating capabilities. One aim is to understand how the two heads of myosin influence each other's function. Molecular biological technique will be used to engineer physiologically relevant "asymmetric" heavy meromyosin constructs in which the two heads of the molecule differ in the state of light chain phosphorylation, in cycling rates, or in actin binding properties. The constructs will be analyzed by biochemical, kinetic (steady-state and transient), and mechanical (velocity and force at the population and single molecule level) techniques. These studies will test existing models for how the two heads interact as well as provide new insight into the positive or negative consequences of having non- identical heads. A second aim is to understand how the actomyosin interface changes as the weak to strong binding transition occurs. The functional impact of mutations at the interface will be analyzed by techniques described above. Image reconstruction of actin decorated withy S1 mutants, in conjunction with docking of the crystal structure, will be used to provide high resolution structural information regarding the actomyosin interface. The third aim is to crystallize a mutant that is arrested in the M.ATP* state to determine if ATP binding or ATP hydrolysis causes the conformational change that results in priming of the lever arm. The overall goal is to understand how the structural domains of myosin contribute to chemomechanical coupling.